Although disulfide bonds are critical to the structure of many secreted proteins, and to the regulation of a range of biochemical processes, their biosynthesis in multicellular organisms remains surprisingly cryptic. Much of this application deals with the poorly-understood Quiescin-sulfhydryl oxidase (QSOX) family of flavoproteins. These vertebrate oxidases introduce disulfide bonds directly into unfolded reduced proteins but have also been identified as growth factors (e.g. bone-derived growth factor, placental-derived prostrate growth factor, and erythroid cell stimulating factor). QSOXs are strongly up-regulated in a number of cancers (most notably of prostrate and bone) and may be involved in the remodeling of the extracellular matrix. The first of six aims of this application explores those factors that make a given protein a good substrate of mammalian QSOX. The second goal is to characterize the nature of QSOX's binding site for protein substrates. A third aim investigates the cooperation between protein disulfide isomerase and QSOX during the oxidative refolding of client proteins. A fourth goal is to express, purify and initiate the first mechanistic study of two additional QSOX family members. Human QSOX2 has been described as a key player in the pathways to apoptosis in neuroblastoma cells and merits a study of enzyme mechanism and substrate specificity. A second intended candidate is recombinant Trypanosoma brucei QSOX. Unlike metazoan QSOXs, their protozoan counterparts substantially lack the second thioredoxin domain and may show important, and exploitable, differences in enzyme mechanism. A fifth aim is a continuation of the design, synthesis and in vitro evaluation of arsenical-based inhibitors of QSOX and related sulfhydryl oxidases. A final goal extends characterization of the smaller sulfhydryl oxidase homolog of QSOX, the flavin-linked augmenter of liver regeneration (ALR). While ALR has a range of important cytokine-like functions in mammals, its intracellular role as a sulfhydryl oxidase, its substrate specificity and its enzymatic reaction mechanism remain poorly understood. Overall, this application is directed towards a better understanding of the redox-enzymology of disulfide generation and isomerization in higher eukaryotes. PUBLIC HEALTH RELEVANCE: This research studies a family of poorly understood enzymes that play diverse roles in protein folding, in the formation and remodeling of the extracellular matrix, and in the regeneration of liver tissue. Some of these proteins are tissue growth factors that are over-expressed in prostrate and bone cancer. A better understanding of the mechanism of these important proteins may help in the design of chemotherapeutic agents.